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. 2013 Aug 1;3(8):1261–1272. doi: 10.1534/g3.113.006213

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Copyright © 2013 Terweij et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Analysis of fluorescent knock-in RITE cassettes. (A) Spc42, a subunit of the spindle pole body, was tagged with a 3xHA→3xT7-mRFP RITE cassette (strain Spc42R). (B) Fluorescent microscopy of strain Spc42R. In the preswitch sample, no mRFP was detected; in the postswitch sample Spc42-mRFP was clearly visible. (C) Immunoblot analysis of the Spc42R samples described in (B). Pgk1 was used as loading control. (D−F) as in (A−C) but using Spc42 tagged with a 1xT7→1xHA-yEGFP RITE cassette (strain Spc42G). Spc42 containing a single HA or T7 tag could not be detected on blots using the tag-specific antibodies. However, an antibody against spacer-LoxP could visualize old and new Spc42. Scale bars, 2 μm.